1. Field of the Invention
The present invention relates to isolated polypeptides having oxaloacetate hydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.
2. Description of the Related Art
Lignin is an aromatic polymer occurring in the woody tissue of higher plants. Due to its hydrophobicity and complex random structure lacking regular hydrolyzable bonds, lignin is poorly degraded by most organisms. The best degraders of lignin are white rot fungi that produce extracellular peroxidases and laccases, which are involved in the initial attack of lignin.
Manganese-dependent peroxidase is a frequently encountered peroxidase produced by white rot fungi (Urzúa et al., 1995, FEBS Letters 371: 132–136). The enzyme catalyzes the oxidation of Mn2+ to Mn3+, which in turn can oxidize residues present in lignin. The peroxidase has a catalytic cycle involving a 2-electron oxidation of its heme by hydrogen peroxide to form compound I. Compound I can be reduced by Mn(II) or a phenolic substrate to compound II, the one electron form of the enzyme. The best reducing substrate for compounds I and II is Mn(II), a metal naturally present in wood. The Mn(III) formed oxidizes other substrates.
Organic acids such as oxalate, glyoxylate and lactate are known to have an important role in the mechanism of manganese-dependent peroxidase and lignin degradation. Mn(III) is stripped from the enzyme by organic acids, and the produced Mn(III)-organic acid complex acts as a diffusible mediator in the oxidation of lignin by manganese-dependent peroxidase. Mn(III) can also oxidize organic acids, yielding radicals. The organic acids may also be supplied from the degradation of lignin and by microorganisms.
Several pathways leading to oxalate have been elucidated. One involves the cleavage of oxaloacetate by oxaloacetate hydrolase to form oxalic acid and acetate (Kubicek et al., 1988, Appl. Environ. Microb. 55: 633–637). Another pathway involves the oxidation of glyoxylate to oxalic acid by the enzyme NAD-glyoxylate dehydrogenase (Balmforth and Thomson, 1984, Biochem. J. 218: 341–342).
WO 2000/50576 describes an Aspergillus niger oxaloacetate hydrolase gene and fungal host cells made deficient in such a gene.
There is a need in the art for improving the lignin degradative ability of strains by increasing the production of enzymes involved in degrading lignin. For example, Ceriporiopsis subvermispora is currently used for biopulping and an engineered strain thereof with an improved lignin degradation capability would be particularly advantageous. Pretreatment of wood chips with Ceriporiopsis subvermispora prior to mechanical pulping has been shown to reduce energy consumption by 30–45% (Messner and Srebotnik 1994, FEMS Microbiology Rev. 13: 351–364; Scott and Swaney, 1998, TAPPI J. 81: 153–175; Scott et al., 2000, Recent Developments in Biopulping Technology at Madison, Wis. In: L Viikari and R Lantto, ed. Progress in Biotechnology 21: Biotechnology in the Pulp and Paper Industry: 8th ICBPPI Meeting. Amsterdam: Elsevier Science B.V. pp 61–71.). While the mechanism(s) of biopulping are unknown, Ceriporiopsis subvermispora colonization of wood appears to involve at least two stages: an initial rapid colonization which softens wood without affecting fiber strength or lignin content, and later an efficient depolymerization and mineralization of lignin (Blanchette et al., 1988, Biomass 15: 93–101; Blanchette, 1991, Annu. Rev. Phytopathol. 29: 381–398; Blanchette, 1994, Can. J. Botany 73: S999–S1010; Blanchette et al., 1997, J. Biotechnology 53: 203–213; Breen and Singleton, 1999, Curr. Opinion Biotech. 10: 252–258).
It is an object of the present invention to provide improved polypeptides having oxaloacetate hydrolase activity and nucleic acids encoding the polypeptides.